雌激素对宫腔粘连纤维化进程及叉头框F2表达的影响

发布时间：2018-09-14 16:10

【摘要】：研究背景:宫腔粘连(IUAs)是指创伤、感染等各种原因所致子宫内膜基底层损伤后,宫腔部分或全部粘连。近年研究表明,子宫内膜纤维化是IUAs的主要病理学特征,是其本质。叉头框F2(FoxF2)是器官发育、细胞外基质合成、上皮-间质细胞转化中的重要转录调节因子,参与了多种器官的纤维化过程。FoxF2在IUAs的作用尚未见文献报道。雌激素是存在于女性体内的最主要性激素,在IUAs治疗中常作为经验性治疗被临床广泛应用,但因疗效不一、基础研究缺乏,其应用剂量仍存在广泛争议。本实验拟探讨FoxF2在IUAs中的表达及不同浓度雌激素对IUAs纤维化进程和FoxF2表达的影响。第一章FoxF2在宫腔粘连细胞模型中的表达及意义目的:探讨FoxF2在IUAs细胞模型中的表达。方法:1.原代培养人子宫内膜基质细胞(HESCs)。2.免疫细胞化学法进行细胞鉴定。3.建立IUAs细胞模型:用0和10ng/ml转化生长因子β1(TGF-β1)分别作用于HESCs 48 h作为对照组和模型组。4.实时荧光定量PCR(qPCR)和蛋白质印迹法(WB)检测α-平滑肌肌动蛋白(α-SMA)、胶原I(COLI)及FoxF2的mRNA和蛋白表达。结果:1.原代培养的HESCs形态稳定,生长活性好。2.免疫细胞化学法检测细胞波形蛋白表达阳性,且波形蛋白阳性细胞比率高,而角蛋白18表达阴性,证实为HESCs,且纯度高。3.与对照组相比,模型组α-SMA、COLI及FoxF2的mRNA和蛋白表达均显著性增高(P0.05)。结论:1.10ng/mlTGF-β1可诱导HESCs向肌成纤维细胞转化,引起纤维化标志物α-SMA和COLI的表达稳定性增加,从而建立IUAs细胞模型。2.FoxF2在IUAs中高表达,提示FoxF2可能参与IUAs纤维化进程。第二章雌激素对宫腔粘连纤维化进程及FoxF2表达的影响目的:探讨不同浓度雌激素对IUAs纤维化进程及FoxF2表达的影响。方法:1.配置10-6、10-8 10-10、10-12mol/L的雌激素工作液。2.本实验分为5组,分别为模型组、10-6mol/L E2组、10-8mol/LE2组、10-10 mol/L E2组、10-12 mol/L E2组。先用 10 ng/ml TGF-β1作用于HESCs 48 h以建立IUAs细胞模型,再用上述不同浓度雌激素作用于该模型中48 h。3.qPCR和WB法检测α-SMA、COLI及FoxF2的mRNA和蛋白表达。结果:1.qPCR 结果:与模型组相比,10-6、10-8、10-10mol/LE2 组 α-SMA、COLI、FoxF2mRNA 表达均下降(10-10mol/LE2组 COLI:P0.05,其余各组P0.05);10-12 mol/LE2组 α-SMA、COLImRNA 表达上升(P0.05),FoxF2mRNA 表达下降(P0.05)。2.WB 结果:与模型组相比,10-6、10-8、10-10 mol/LE2组 α-SMA、COLI、FoxF2蛋白表达均下降(P0.05);10-12 mol/LE2组α-SMA蛋白表达上升(P0.05),COLI和FoxF2蛋白表达均下降(P0.05)。结论:17β-雌二醇可在一定范围内下调α-SMA和COLI的表达,逆转IUAs纤维化进程,并抑制FoxF2的表达。
[Abstract]:Background: intrauterine adhesions (IUAs) refer to partial or total adhesions of uterine cavity after endometrial basal layer injury caused by trauma and infection. Recent studies have shown that endometrial fibrosis is the main pathological feature and essence of IUAs. Forked frame F2 (FoxF2) is an important transcriptional regulator in organ development, extracellular matrix synthesis and epithelial-interstitial cell transformation. The role of FoxF2 in the process of organ fibrosis has not been reported in the literature. Estrogen is the most important sex hormone in women. It is often used as empirical therapy in IUAs. However, because of the difference of curative effect and lack of basic research, the dosage of estrogen is still controversial. The purpose of this study was to investigate the expression of FoxF2 in IUAs and the effects of different concentrations of estrogen on the progression of IUAs fibrosis and the expression of FoxF2. The expression and significance of FoxF2 in uterine Adhesion Cell Model objective: to investigate the expression of FoxF2 in IUAs cell model. Method 1: 1. Primary culture of human endometrial stromal cells (HESCs). 2. Immunocytochemistry method for cell identification. IUAs cell model was established: 0 and 10ng/ml transforming growth factor 尾 1 (TGF- 尾 1) were treated with HESCs for 48 h as control group and model group respectively. The expression of 伪 -smooth muscle actin (伪 -SMA), collagen I (COLI) and FoxF2 was detected by real-time quantitative PCR (qPCR) and Western blot (WB). The result is 1: 1. The primary culture of HESCs was stable in morphology and good in growth activity. 2. The positive expression of vimentin was detected by immunocytochemistry, and the positive rate of vimentin was high, but the expression of keratin 18 was negative, which was proved to be HESCs, with high purity. 3. Compared with the control group, the mRNA and protein expressions of 伪 -SMA-COLI and FoxF2 in the model group were significantly higher than those in the control group (P0.05). Conclusion: 1. 10 ng / ml TGF- 尾 1 can induce the transformation of HESCs to myofibroblasts and increase the stability of 伪 -SMA and COLI expression, thus establishing the IUAs cell model. 2. FoxF2 is highly expressed in IUAs, suggesting that FoxF2 may participate in the process of IUAs fibrosis. The effect of estrogen on the process of intrauterine adhesion fibrosis and the expression of FoxF2 objective: to investigate the effects of different concentrations of estrogen on the progression of IUAs fibrosis and the expression of FoxF2. Method 1: 1. Oestrogen working solution. 2 with 10-6, 10-8, 10-10, 10-12 mol / L, 10-8, 10-10, 10-12 mol / L. The experiment was divided into five groups: model group 10-6 mol / L E2 group 10-8 mol / L E _ 2 group 10-10 mol/L E _ 2 group 10-12 mol/L E _ 2 group. The IUAs cell model was established by 10 ng/ml TGF- 尾 1 treatment with HESCs for 48 h. The mRNA and protein expressions of 伪 -SMA-COLI and FoxF2 were detected by 48 h.3.qPCR and WB methods. Results 1. QPCR results: compared with the model group, the expression of 伪 -SMACOLICOLI FoxF2 mRNA in 10-6 + 10-8 10-10 mol / L 2 group decreased (P0.05) in 10-12 mol/LE2 group (P0.05). 2.WB results: compared with the model group, the expression of 伪 -SMACOLICOLI FoxF2 protein in 10-6 10-8 10-10 mol/LE2 group decreased (P0.05) in 10-12 mol/LE2 group (P0.05). 2. WB results: compared with the model group, the expression of 伪 -SMACOLICOLICOLI FoxF2 protein in 10-12 mol/LE2 group was decreased (P0.05), and the expression of 伪 -SMACOLICOLI FoxF2 protein in 10-12 mol/LE2 group was significantly decreased compared with the model group (P0.05). The expression of coli and FoxF2 decreased significantly (P0.05). Conclusion the expression of 伪 -SMA and COLI can be down-regulated by 尾 -estradiol in a certain range, which can reverse the process of IUAs fibrosis and inhibit the expression of FoxF2.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.74

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